Human pancreatic ribonuclease (RNase 1) is an enzyme that can block the flow of biochemical information by catalyzing the cleavage of cellular RNA. Our intent is to use ideas and methods from enzymology, biochemistry and molecular biology, cell biology, and molecular genetics to reveal attributes of the structure and function of RNase 1 that can lead to toxic activity against cancer cells, to enhance that activity, and to reveal fundamental information on the role of RNase 1 in mammalian biology. During the next grant period, this intent will be achieved in five Specific Aims. Specific Aims: In Aim 1, we shall identify specific glycans on the surface of human cells that interact with RNase 1, characterize those interactions, and assess their biological relevance. In Aim 2, we shall develop small-molecule fluorogenic probes for two critical steps in RNase 1-mediated cytotoxicity: endocytosis and endosomal escape. In Aim 3, we shall determine the fate of cellular RNA exposed to an RI-evasive variant of RNase 1, and whether the products of catalysis by RNase 1 are substrates for a cytosolic enzyme, RtcB. In Aim 4, we shall produce RNase 1 with human-like glycosylation and determine the effect of the glycans on the biochemical and biological attributes of the enzyme. In Aim 5, we shall create mice in which the gene that encodes the mouse homolog of RNase 1 has been disrupted in a conditional manner. We shall characterize the phenotype of these mice so as to reveal the role(s) of RNase 1 in a mammal. Significance: The results of the research proposed herein will provide a detailed biochemical understanding of the antitumor activity of RNase 1, and could ultimately lead to new chemotherapeutic agents based on variants of an endogenous human enzyme.